Kiel Hards, Duncan G. G. McMillan, Lici A. Schurig-Briccio, Robert B. Gennis, Holger Lill, Dirk Bald, and Gregory M. Cook
PNAS, June 25, 2018. https://doi.org/10.1073/pnas.1803723115
Bedaquiline (BDQ), an inhibitor of the mycobacterial F1Fo-ATP synthase, has revolutionized the antitubercular drug discovery program by defining energy metabolism as a potent new target space. Several studies have recently suggested that BDQ ultimately causes mycobacterial cell death through a phenomenon known as uncoupling. The biochemical basis underlying this, in BDQ, is unresolved and may represent a new pathway to the development of effective therapeutics. In this communication, we demonstrate that BDQ can inhibit ATP synthesis in Escherichia coli by functioning as a H+/K+ ionophore, causing transmembrane pH and potassium gradients to be equilibrated. Despite the apparent lack of a BDQ-binding site, incorporating the E. coli Fo subunit into liposomes enhanced the ionophoric activity of BDQ. We discuss the possibility that localization of BDQ at F1Fo-ATP synthases enables BDQ to create an uncoupled microenvironment, by antiporting H+/K+. Ionophoric properties may be desirable in high-affinity antimicrobials targeting integral membrane proteins.
Antibiotics generally target one of five essential cellular functions in bacteria, but many of these targets are now compromised through rapidly spreading antibiotic resistance. Bedaquiline (BDQ), a new FDA-approved antitubercular drug, targets energy metabolism: defining cellular energetics as a new target space for antibiotics. This is a relatively unexplored area, as BDQ was only FDA approved in 2012. Several studies have recently found that BDQ stimulates mycobacterial respiration, in addition to inhibiting its molecular target, the F1Fo-ATP synthase. We show that BDQ is an ionophore, which shuttles H+ and K+ ions across membranes, and propose that this activity may contribute to killing of mycobacteria by BDQ. Combining ionophoric activity with high-affinity membrane protein inhibition may enhance the specificity and potency of antibiotics.
Pia Steigler, Naomi J. Daniels, Tim R. McCulloch, Brin M. Ryder, Sarah K. Sandford and Joanna R. Kirman
Immunology and Cell Biology 2018; 96: 379-389. https://doi.org/10.1111/imcb.12007
The tuberculosis (TB) vaccine bacille Calmette–Guérin (BCG) prevents disseminated childhood TB; however, it fails to protect against the more prevalent pulmonary TB. Limited understanding of the immune response to Mycobacterium tuberculosis, the causative agent of TB, has hindered development of improved vaccines. Although memory CD4 T cells are considered the main mediators of protection against TB, recent studies suggest there are other key subsets that contribute to antimycobacterial immunity. To that end, innate cells may be involved in the protective response. In this study, we investigated the primary response of innate lymphoid cells (ILCs) to BCG exposure. Using a murine model, we showed that ILCs increased in number in the lungs and lymph nodes in response to BCG vaccination. Additionally, there was significant production of the antimycobacterial cytokine IFN‐γ by ILCs. As ILCs are located at mucosal sites, it was investigated whether mucosal vaccination (intranasal) stimulated an enhanced response compared to the traditional vaccination approach (intradermal or subcutaneous). Indeed, in response to intranasal vaccination, the number of ILCs, and IFN‐γ production in NK cells and ILC1s in the lungs and lymph nodes, were higher than that provoked through intradermal or subcutaneous vaccination. This work provides the first evidence that BCG vaccination activates ILCs, paving the way for future research to elucidate the protective potential of ILCs against mycobacterial infection. Additionally, the finding that lung ILCs respond rigorously to mucosal vaccination may have implications for the delivery of novel TB vaccines.
The first author, Pia Steigler, completed her PhD in Jo Kirman's lab. Naomi Daniels is a Postdoctoral Fellow, Sarah Sandford is Research Assistant, and Brin Ryder is a current PhD student. Tim McCulloch is at the Walter and Eliza Hall Institute of Medical Research in Victoria, Australia.
Rachel Kaminsky and Sergio E. Morales
Frontiers in Microbiology 9:809. https://doi.org/10.3389/fmicb.2018.00809
The rare biosphere is predicted to aid in maintaining functional redundancy as well as contributing to community turnover across many environments. Recent developments have partially confirmed these hypotheses, while also giving new insights into dormancy and activity among rare communities. However, less attention has been paid to the rare biosphere in soils. This study provides insight into the rare biosphere’s contribution to soil microbial diversity through the study of 781 soil samples representing 24 edaphically diverse sites. Results show that Bray–Curtis dissimilarity for time-sensitive conditionally rare taxa (CRT) does not correlate with whole community dissimilarity, while dissimilarity for space-sensitive CRT only weakly correlate with whole community dissimilarity. This adds to current understanding of spatiotemporal filtering of rare taxa, showing that CRT do not account for community variance across tested soils, but are under the same selective pressure as the whole community.
Rachel Kaminsky is a PhD student in Dr Sergio Morales' lab.
Hannah G. Hampton, Simon A. Jackson, Robert D. Fagerlund, Anne I.M. Vogel, Ron L. Dy, Tim R. Blower, Peter C. Fineran
Journal of Molecular Biology, Volume 430, Issue 8, 13 April 2018, Pages 1141-1156
Bacteria resist phage infection using multiple strategies, including CRISPR-Cas and abortive infection (Abi) systems. Abi systems provide population-level protection from phage predation, via “altruistic” cell suicide. It has recently been shown that some Abi systems function via a toxin–antitoxin mechanism, such as the widespread AbiE family. The Streptococcus agalactiae AbiE system consists of a bicistronic operon encoding the AbiEi antitoxin and AbiEii toxin, which function as a Type IV toxin–antitoxin system. Here we examine the AbiEi antitoxin, which belongs to a large family of transcriptional regulators with a conserved N-terminal winged helix-turn-helix domain. This winged helix-turn-helix is essential for transcriptional repression of the abiE operon. The function of the AbiEi C-terminal domain is poorly characterized, but it contributes to transcriptional repression and is sufficient for toxin neutralization. We demonstrate that a conserved charged surface on one face of the C-terminal domain assists sequence-specific DNA binding and negative autoregulation, without influencing antitoxicity. Furthermore, AbiEi binds cooperatively to two inverted repeats within the abiE promoter and bends the DNA by 72°. These findings demonstrate that the mechanism of DNA binding by the widespread family of AbiEi antitoxins and transcriptional regulators can contribute to negative autoregulation.
Hannah Hampton is a PhD student in Peter Fineran's lab. Simon Jackson and Robert Fagerlund are Postdoctoral Fellows.
Bridget N.J. Watson, Raymond H.J. Staals, Peter C. Fineran
mBio, January/February 2018, Volume 9, Issue 1
A powerful contributor to prokaryotic evolution is horizontal gene transfer (HGT) through transformation, conjugation, and transduction, which can be advantageous, neutral, or detrimental to fitness. Bacteria and archaea control HGT and phage infection through CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) adaptive immunity. Although the benefits of resisting phage infection are evident, this can come at a cost of inhibiting the acquisition of other beneficial genes through HGT. Despite the ability of CRISPR-Cas to limit HGT through conjugation and transformation, its role in transduction is largely overlooked. Transduction is the phage-mediated transfer of bacterial DNA between cells and arguably has the greatest impact on HGT. We demonstrate that in Pectobacterium atrosepticum, CRISPR-Cas can inhibit the transduction of plasmids and chromosomal loci. In addition, we detected phage-mediated transfer of a large plant pathogenicity genomic island and show that CRISPR-Cas can inhibit its transduction. Despite these inhibitory effects of CRISPR-Cas on transduction, its more common role in phage resistance promotes rather than diminishes HGT via transduction by protecting bacteria from phage infection. This protective effect can also increase transduction of phage-sensitive members of mixed populations. CRISPR-Cas systems themselves display evidence of HGT, but little is known about their lateral dissemination between bacteria and whether transduction can contribute. We show that, through transduction, bacteria can acquire an entire chromosomal CRISPR-Cas system, including cas genes and phage-targeting spacers. We propose that the positive effect of CRISPR-Cas phage immunity on enhancing transduction surpasses the rarer cases where gene flow by transduction is restricted.
The generation of genetic diversity through acquisition of DNA is a powerful contributor to microbial evolution and occurs through transformation, conjugation, and transduction. Of these, transduction, the phage-mediated transfer of bacterial DNA, is arguably the major route for genetic exchange. CRISPR-Cas adaptive immune systems control gene transfer by conjugation and transformation, but transduction has been mostly overlooked. Our results indicate that CRISPR-Cas can impede, but typically enhances the transduction of plasmids, chromosomal genes, and pathogenicity islands. By limiting wild-type phage replication, CRISPR-Cas immunity increases transduction in both phage-resistant and -sensitive members of mixed populations. Furthermore, we demonstrate mobilization of a chromosomal CRISPR-Cas system containing phage-targeting spacers by generalized transduction, which might partly account for the uneven distribution of these systems in nature. Overall, the ability of CRISPR-Cas to promote transduction reveals an unexpected impact of adaptive immunity on horizontal gene transfer, with broader implications for microbial evolution.
Bridget Watson is a PhD student in Peter Fineran's lab and Raymond Staals is a former Postdoctoral Fellow.
The paper was also highlighted in Nature Reviews Microbiology.
Mike Strauss, Nadishka Jayawardena, Eileen Sun, Richard A. Easingwood, Laura N. Burga, Mihnea Bostina
Seneca Valley virus (SVV), like some other members of the Picornaviridae, forms naturally occurring empty capsids, known as procapsids. Procapsids have the same antigenicity as full virions, so they present an interesting possibility for the formation of stable virus-like particles. Interestingly, although SVV is a livestock pathogen, it has also been found to preferentially infect tumor cells and is being explored for use as a therapeutic agent in the treatment of small-cell lung cancers. Here we used cryo-electron microscopy to investigate the procapsid structure and describe the transition of capsid protein VP0 to the cleaved forms of VP4 and VP2. We show that the SVV receptor binds the procapsid, as evidence of its native antigenicity. In comparing the procapsid structure to that of the full virion, we also show that a cage of RNA serves to stabilize the inside surface of the virus, thereby making it more acid stable.
Viruses are extensively studied to help us understand infection and disease. One of the by-products of some virus infections are the naturally occurring empty virus capsids (containing no genome), termed procapsids, whose function remains unclear. Here we investigate the structure and formation of the procapsids of Seneca Valley virus, to better understand how they form, what causes them to form, how they behave, and how we can make use of them. One potential benefit of this work is the modification of the procapsid to develop it for targeted in vivo delivery of therapeutics or to make a stable vaccine against SVV, which could be of great interest to the agricultural industry.
Nadishka Jayawardena is a second-year PhD student and Laura Burga is a Research Fellow, both based in Mihnea Bostina's lab.
As first-equal author, Nadishka performed all the wet-lab experiments, analysed structure and prepared all the figures for the paper (54 panels, over 9 figures). The paper was selected by the editors for Spotlight, a feature in which a handful of articles of special importance are selected from around 50 published in each issue. He produced the below structural image.
Sergio E. Morales, Blandine Trouche, Steve Wakelin, Murom Banabas, Paul N. Nelson
Applied Soil Ecology https://doi.org/10.1016/j.apsoil.2017.11.028
The Anthropocene is linked to massive land use changes as a result of human activity. While aboveground changes in biodiversity are well documented, the effects on belowground microbial communities are less understood, yet could impact on many ecosystem functions. Here we aimed to identify differences in belowground microbial diversity between forest and grassland sites in a humid tropical mosaic landscape in Papua New Guinea. Using DNA-based amplicon sequencing targeting the 16S rRNA gene, prokaryotic community composition was assessed from surface soil samples. The composition of prokaryotic communities (beta diversity) differed between forest and grassland sites despite maintaining similar richness (alpha diversity) levels. Changes in community structure were small at higher taxonomic levels, but strong at the operational taxonomic unit (OTU) level but for a small subset of taxa. Changes in community composition between sites (based on Bray-Curtis distance) reflected a large rearrangement with species assemblage (OTU) differing by 68%. The results suggest that ecosystem change in this landscape leads to ecological filtering and selection at lower, but not higher taxonomic levels.
Dr Sergio Morales is a Senior Lecturer in the Department of Microbiology and Immunology. Blandine Trouche was a visiting student in Dr Morales' lab at the time the work was undertaken.
Varakorn Kosaisavee, Rossarin Suwanarusk, Adeline C. Y. Chua, Dennis E. Kyle, Benoit Malleret, Rou Zhang, Mallika Imwong, Rawiwan Imerbsin, Ratawan Ubalee, Hugo Sámano-Sánchez, Bryan K. S. Yeung, Jessica J. Y. Ong, Eric Lombardini, François Nosten, Kevin S. W. Tan, Pablo Bifani, Georges Snounou, Laurent Rénia, Bruce Russell
The corresponding author, Dr Bruce Russell, is a researcher in the Department of Microbiology and Immunology. Dr Rossarin Suwanarusk is a Research Fellow in the Russell Lab, while Adeline Chua and Jessica Ong are PhD students.
Sarah C. Saunderson and Alexander D. McLellan
Journal of Immunology October 1, 2017, 199(7) 2225-2235; DOI: https://doi.org/10.4049/jimmunol.1601537
Exosomes are lipid nanovesicles released after fusion of the endosomal limiting membrane with the plasma membrane. In this study, we investigated the requirement for CD4 T cells, B cells, and NK cells to provide help for CD8 T cell–mediated response to B cell–derived exosomes. CTL responses to Ag-loaded exosomes were dependent on host MHC class I, with a critical role for splenic langerin+ CD8α+ dendritic cells (DCs) in exosomal Ag cross-presentation. In addition, there was an absolute dependence on the presence of CD4 T cells, CD8 T cells, and NK cells, where the loss of any one of these subsets led to a complete loss of CTL response. Interestingly, NK cell depletion experiments demonstrated a critical cutoff point for depletion efficacy, with low-level residual NK cells providing sufficient help to allow optimal CD8 T cell proliferative responses to exosomal protein. Despite the potential role for B cells in the response to B cell–derived exosomal proteins, B cell depletion did not alter the exosome-induced CTL response. Similarly, a possible role for the BCR or circulating Ab in mediating CTL responses to B cell–derived exosomes was ruled out using DHLMP2A mice, which lack secreted and membrane-bound Ab, yet harbor marginal zone and follicular B cells. In contrast, CTL responses to DC-derived exosomes were significantly inhibited within Ab-deficient DHLMP2A mice compared with wild-type mice. However, this response was not restored upon serum transfer, implicating a role for the BCR, but not circulating Ab, in DC-derived exosome responses.
Associate Professor Alexander McLellan is an Immunologist in the Department of Microbiology and Immunology, and Dr Sarah Saunderson is a Postdoctoral Fellow.
Leila J. Nicholson, Jackie E. Mahar, Tanja Strive, Tao Zheng, Edward C. Holmes, Vernon K. Ward and Janine A. Duckworth
Applied and Environmental Microbiology, June 2017 vol. 83 no. 11
The Czech v351 strain of rabbit hemorrhagic disease virus (RHDV1) is used in Australia and New Zealand as a biological control agent for rabbits, which are important and damaging introduced vertebrate pests in these countries. However, nonpathogenic rabbit caliciviruses (RCVs) can provide partial immunological cross-protection against lethal RHDV infection and thus interfere with effective rabbit biocontrol. Antibodies that cross-reacted against RHDV antigens were found in wild rabbits before the release of RHDV1 in New Zealand in 1997, suggesting that nonpathogenic RCVs were already present in New Zealand. The aim of this study was to confirm the presence of nonpathogenic RCV in New Zealand and describe its geographical distribution. RCV and RHDV antibody assays were used to screen serum samples from 350 wild rabbits from 14 locations in New Zealand. The serological survey indicated that both RCV and RHDV are widespread in New Zealand wild rabbits, with antibodies detected in 10 out of 14 and 12 out of 14 populations, respectively. Two closely related RCV strains were identified in the duodenal tissue from a New Zealand wild rabbit (RCV Gore-425A and RCV Gore-425B). Both variants are most closely related to Australian RCV strains, but with 88% nucleotide identity, they are genetically distinct. Phylogenetic analysis revealed that the New Zealand RCV strains fall within the genetic diversity of the Australian RCV isolates, indicating a relatively recent movement of RCVs between Australia and New Zealand.
Wild rabbits are important and damaging introduced vertebrate pests in Australia and New Zealand. Although RHDV1 is used as a biological control agent, some nonpathogenic RCVs can provide partial immunological cross-protection against lethal RHDV infection and thus interfere with its effectiveness for rabbit control. The presence of nonpathogenic RCVs in New Zealand wild rabbits has been long hypothesized, but earlier attempts to isolate a New Zealand RCV strain have been unsuccessful. Therefore, it is important to determine if such nonpathogenic viruses exist in New Zealand rabbits, especially considering the proposed introduction of new RHDV strains into New Zealand as biocontrols.
Prashanth T. Nagesh, Mazhar Hussain, Henry D. Galvin and Matloob Husain
Frontiers in Microbiology 2017; 8: 1315. doi: 10.3389/fmicb.2017.01315
Host cells produce variety of antiviral factors that create an antiviral state and target various stages of influenza A virus (IAV) life cycle to inhibit infection. However, IAV has evolved various strategies to antagonize those antiviral factors. Recently, we reported that a member of class I host histone deacetylases (HDACs), HDAC1 possesses an anti-IAV function. Herein, we provide evidence that HDAC2, another class I member and closely related to HDAC1 in structure and function, also possesses anti-IAV properties. In turn, IAV, like HDAC1, dysregulates HDAC2, mainly at the polypeptide level through proteasomal degradation to potentially minimize its antiviral effect. We found that IAV downregulated the HDAC2 polypeptide level in A549 cells in an H1N1 strain-independent manner by up to 47%, which was recovered to almost 100% level in the presence of proteasome-inhibitor MG132. A further knockdown in HDAC2 expression by up to 90% via RNA interference augmented the growth kinetics of IAV in A549 cells by more than four-fold after 24 h of infection. Furthermore, the knockdown of HDAC2 expression decreased the IAV-induced phosphorylation of the transcription factor, Signal Transducer and Activator of Transcription I (STAT1) and the expression of interferon-stimulated gene, viperin in infected cells by 41 and 53%, respectively. The role of HDAC2 in viperin expression was analogous to that of HDAC1, but it was not in the phosphorylation of STAT1. This indicated that, like HDAC1, HDAC2 is a component of IAV-induced host innate antiviral response and performs both redundant and non-redundant functions vis-a-vis HDAC1; however, IAV dysregulates them both in a redundant manner.
Robert D. Fagerlund, Max E. Wilkinson, Oleg Klykov, Arjan Barendregt, Grant Pearce, Sebastian N. Kieper, Howard W. R. Maxwell, Angela Capolupo, Albert J. R. Heck, Kurt L. Krause, Mihnea Bostina, Richard A. Scheltema, Raymond H. J. Staals, and Peter C. Fineran
PNAS, vol. 114 no. 26, E5122–E5128, doi: 10.1073/pnas.1618421114
CRISPR-Cas adaptive immune systems capture DNA fragments from invading bacteriophages and plasmids and integrate them as spacers into bacterial CRISPR arrays. In type I-E and II-A CRISPR-Cas systems, this adaptation process is driven by Cas1–Cas2 complexes. Type I-F systems, however, contain a unique fusion of Cas2, with the type I effector helicase and nuclease for invader destruction, Cas3. By using biochemical, structural, and biophysical methods, we present a structural model of the 400-kDa Cas14–Cas2-32 complex from Pectobacterium atrosepticum with bound protospacer substrate DNA. Two Cas1 dimers assemble on a Cas2 domain dimeric core, which is flanked by two Cas3 domains forming a groove where the protospacer binds to Cas1–Cas2. We developed a sensitive in vitro assay and demonstrated that Cas1–Cas2-3 catalyzed spacer integration into CRISPR arrays. The integrase domain of Cas1 was necessary, whereas integration was independent of the helicase or nuclease activities of Cas3. Integration required at least partially duplex protospacers with free 3′-OH groups, and leader-proximal integration was stimulated by integration host factor. In a coupled capture and integration assay, Cas1–Cas2-3 processed and integrated protospacers independent of Cas3 activity. These results provide insight into the structure of protospacer-bound type I Cas1–Cas2-3 adaptation complexes and their integration mechanism.
Manmeet Bhalla, Daria Law, Georgina C. Dowd and Keith Ireton
American Society for Microbiology Infection and Immunity, 1 May 2017, doi:10.1128/IAI.00087-17
The bacterial pathogen Listeria monocytogenes causes food-borne illnesses resulting in gastroenteritis, meningitis, or abortion. Listeria induces its internalization into some human cells through interaction of the bacterial surface protein InlB with the host receptor tyrosine kinase Met. InlB-dependent entry requires localized polymerization of the host actin cytoskeleton. Signal transduction pathways that act downstream of Met to regulate actin filament assembly or other processes during Listeria uptake remain incompletely characterized. Here, we demonstrate important roles for the human serine/threonine kinases mTOR and Protein Kinase C (PKC)-α in InlB-dependent entry. Experiments involving RNA interference (RNAi) indicated that two multi-protein complexes containing mTOR, mTORC1 and mTORC2, are each needed for efficient internalization of Listeria into the human cell line HeLa. InlB stimulated Met-dependent phosphorylation of mTORC1 or mTORC2 substrates, demonstrating activation of both mTOR-containing complexes. RNAi studies indicated that the mTORC1 effectors 4E-BP1 and HIF-1α and the mTORC2 substrate Protein Kinase C (PKC)-α each control Listeria uptake. Genetic or pharmacological inhibition of PKC-α reduced internalization of Listeria and accumulation of actin filaments that normally accompanies InlB-mediated entry. Collectively, our results identify mTOR and PKC-α as host factors exploited by Listeria to promote infection. PKC-α controls Listeria entry, at least in part, by regulating the actin cytoskeleton downstream of the Met receptor
Blair Lawley, Karen Munro, Alan Hughes, Alison J. Hodgkinson, Colin G. Prosser, Dianne Lowry, Shao J. Zhou, Maria Makrides, Robert A. Gibson, Christophe Lay, Charmaine Chew, Pheng Soon Lee, Khai Hong Wong and Gerald W. Tannock.
PeerJ 5:e3375; DOI 10.7717/peerj.3375
Members of the genus Bifidobacterium are abundant in the feces of babies during the exclusively-milk-diet period of life. Bifidobacterium longum is reported to be a common member of the infant fecal microbiota. However, B. longum is composed of three subspecies, two of which are represented in the bowel microbiota (B. longum subsp. longum; B. longum subsp. infantis). B. longum subspecies are not differentiated in many studies, so that their prevalence and relative abundances are not accurately known. This may largely be due to difficulty in assigning subspecies identity using DNA sequences of 16S rRNA or tuf genes that are commonly used in bacterial taxonomy.
We developed a qPCR method targeting the sialidase gene (subsp. infantis) and sugar kinase gene (subsp. longum) to differentiate the subspecies using specific primers and probes. Specificity of the primers/probes was tested by in silico, pangenomic search, and using DNA from standard cultures of bifidobacterial species. The utility of the method was further examined using DNA from feces that had been collected from infants inhabiting various geographical regions.
A pangenomic search of the NCBI genomic database showed that the PCR primers/probes targeted only the respective genes of the two subspecies. The primers/probes showed total specificity when tested against DNA extracted from the gold standard strains (type cultures) of bifidobacterial species detected in infant feces. Use of the qPCR method with DNA extracted from the feces of infants of different ages, delivery method and nutrition, showed that subsp. infantis was detectable (0–32.4% prevalence) in the feces of Australian (n = 90), South-East Asian (n = 24), and Chinese babies (n = 91), but in all cases at low abundance (<0.01–4.6%) compared to subsp. longum (0.1–33.7% abundance; 21.4–100% prevalence). Discussion. Our qPCR method differentiates B. longum subspecies longum and infantis using characteristic functional genes. It can be used as an identification aid for isolates of bifidobacteria, as well as in determining prevalence and abundance of the subspecies in feces. The method should thus be useful in ecological studies of the infant gut microbiota during early life where an understanding of the ecology of bifidobacterial species may be important in developing interventions to promote infant health.
The lead author, Blair Lawley, is a Scientific Officer in Professor Gerald Tannock's lab. Alan Hughes is research staff in the Cook Lab. The work was undertaken with the assistance of researchers in New Zealand, Australia and Singapore.
Saeed Sharif, Yoshio Nakatani, Lyn Wise, Michael Corbett, Nicola C. Real, Gabriella S. Stuart, Zabeen Lateef, Kurt Krause, Andrew A. Mercer, Stephen B. Fleming
Bovine papular stomatitis virus (BPSV) is a Parapoxvirus that induces acute pustular skin lesions in cattle and is transmissible to humans. Previous studies have shown that BPSV encodes a distinctive chemokine-binding protein (CBP). Chemokines are critically involved in the trafficking of immune cells to sites of inflammation and infected tissue, suggesting that the CBP plays a role in immune evasion by preventing immune cells reaching sites of infection. We hypothesised that the BPSV-CBP binds a wide range of inflammatory chemokines particularly those involved in BPSV skin infection, and inhibits the recruitment of immune cells from the blood into inflamed skin. Molecular analysis of the purified protein revealed that the BPSV-CBP is a homodimeric polypeptide with a MW of 82.4 kDa whilst a comprehensive screen of inflammatory chemokines by surface plasmon resonance showed high-affinity binding to a range of chemokines within the CXC, CC and XC subfamilies. Structural analysis of BPSV-CBP, based on the crystal structure of orf virus CBP, provided a probable explanation for these chemokine specificities at a molecular level. Functional analysis of the BPSV-CBP using transwell migration assays demonstrated that it potently inhibited chemotaxis of murine neutrophils and monocytes in response to CXCL1, CXCL2 as well as CCL2, CCL3 and CCL5 chemokines. In order to examine the effects of CBP in vivo, we used murine skin models to determine its impact on inflammatory cell recruitment such as that observed during BPSV infection. Intradermal injection of BPSV-CBP blocked the influx of neutrophils and monocytes in murine skin in which inflammation was induced with lipopolysaccharide. Furthermore, intradermal injection of BPSV-CBP into injured skin, which more closely mimics BPSV lesions, delayed the influx of neutrophils and reduced the recruitment of MHC-II+ immune cells to the wound bed. Our findings suggest that the CBP could be important in pathogenesis of BPSV infections.
Lead author Saeed Sharif is a PhD student in the Department of Microbiology and Immunology, and produced this work with a support of a group of colleagues from the department. Prpfessor Kurt Krause is based in the neighbouring Department of Biochemistry. The research was supported by funding from the Health Research Council of New Zealand and the University of Otago Special Health Research Scholarship.
Rachel Kaminsky, Blandine Trouche and Sergio E. Morales
Scientific Reports, 2017; 7: 45369. Published online 2017 Mar 28. doi:10.1038/srep45369
Agricultural land is typically managed based on visible plant life at the expense of the belowground majority. However, microorganisms mediate processes sustaining plant life and the soil environment. To understand the role of microbes we first must understand what controls soil microbial community assembly. We assessed the distribution and composition of prokaryotic communities from soils representing four geographic regions on the South Island of New Zealand. These soils are under three different uses (dairy, sheep and beef, and high country farming) and are representative of major soil classification groups (brown, pallic, gley and recent). We hypothesized that pH would account for major community patterns based on 16S profiles, but that land use and location would be secondary modifiers. Community diversity and structure was linked to pH, coinciding with land use. Soil classification correlated with microbial community structure and evenness, but not richness in high country and sheep and beef communities. The impact of land use and pH remained significant at the regional scale, but soil classification provided support for community variability not explained by either of those factors. These results suggest that several edaphic properties must be examined at multiple spatial scales to robustly examine soil prokaryotic communities.
Lead author Rachel Kaminsky is a PhD student in the Department of Microbiology and Immunology. She worked with the help of supervisor Dr Sergio Morales and fellow student Blandine Trouche. This work was funded in part by a grant from fertiliser company Mainland Minerals Ltd.
Journal of Virology, January 2017 vol. 91no. 1 e01430-16
Hypoxia-inducible factor (HIF) is a transcriptional activator with a central role in regulating cellular responses to hypoxia. It is also emerging as a major target for viral manipulation of the cellular environment. Under normoxic conditions, HIF is tightly suppressed by the activity of oxygen-dependent prolyl and asparaginyl hydroxylases. The asparaginyl hydroxylase active against HIF, factor inhibiting HIF (FIH), has also been shown to hydroxylate some ankyrin repeat (ANK) proteins. Using bioinformatic analysis, we identified the five ANK proteins of the parapoxvirus orf virus (ORFV) as potential substrates of FIH. Consistent with this prediction, coimmunoprecipitation of FIH was detected with each of the ORFV ANK proteins, and for one representative ORFV ANK protein, the interaction was shown to be dependent on the ANK domain. Immunofluorescence studies revealed colocalization of FIH and the viral ANK proteins. In addition, mass spectrometry confirmed that three of the five ORFV ANK proteins are efficiently hydroxylated by FIH in vitro. While FIH levels were unaffected by ORFV infection, transient expression of each of the ORFV ANK proteins resulted in derepression of HIF-1α activity in reporter gene assays. Furthermore, ORFV-infected cells showed upregulated HIF target gene expression. Our data suggest that sequestration of FIH by ORFV ANK proteins leads to derepression of HIF activity. These findings reveal a previously unknown mechanism of viral activation of HIF that may extend to other members of the poxvirus family.
The people involved
Da-Yuan Chen recently completed a Phd in Virology with the Virus Research Unit (VRU), Department of Microbiology and Immunology. Colleagues include Dr Stephen Fleming and Professor Andrew Mercer, also of the VRU. Additional assistance was provided by researchers from the University of Adelaide, the QIMR Berghofer Medical Research Institute (Queensland) and Flinders University.
Morad-Rémy Muhsin-Sharafaldine, Bailey R. Kennedy, Sarah C. Saunderson, Catrin R. Buchanan, Amy C. Dunn, James M. Faed, Alexander D. McLellan
Biochimica et Biophysica Acta (BBA) - General Subjects, Volume 1861, Issue 2, February 2017
Chemotherapy induces the release of apoptotic vesicles (ApoV) from the tumor plasma membrane. Tumor ApoV may enhance the risk of thrombotic events in cancer patients undergoing chemotherapy. However, the relative contribution of ApoV to coagulation and the pathways involved remain poorly characterized. In addition, this study sets out to compare the procoagulant activity of chemotherapy-induced ApoV with their cell of origin and to determine the mechanisms of ApoV-induced coagulation.
We utilized human and murine cancer cell lines and chemotherapeutic agents to determine the requirement for the coagulation factors (tissue factor; TF, FII, FV, FVII, FVIII, FIX and phosphatidylserine) in the procoagulant activity of ApoV. The role of previously identified ApoV-associated FV was determined in a FV functional assay.
ApoV were significantly more procoagulant per microgram of protein compared to parental living or dying tumor cells. In the phase to peak fibrin generation, procoagulant activity was dependent on phosphatidylserine, TF expression, FVII and the prothrombinase complex. However, the intrinsic coagulation factors FIX and FVIII were dispensable. ApoV-associated FV could not support coagulation in the absence of supplied, exogenous FV.
ApoV are significantly more procoagulant than their parental tumor cells. ApoV require the extrinsic tenase and prothrombinase complex to activate the early phase of coagulation. Endogenous FV identified on tumor ApoV is serum-derived and functional, but is non-essential for ApoV-mediated fibrin generation.
Morad-Rémy Muhsin-Sharafaldine and Bailey Kennedy are PhD students under the supervision of Associate Professor Alex McLellan. Their colleagues Dr Sarah Saunderson, Catrin Buchanan and Amy Dunn are a Postdoctoral Fellow, former student and Technician from the same lab. Dr James Faed is based in the University of Otago's Department of Pathology.
Manuela Centanni, Jennifer C. Hutchison, Susan M. Carnachan, Alison M. Daines, William J. Kelly, Gerald W. Tannock, Ian M. Sims
Carbohydrate Polymers, Volume 157, 10 February 2017, Pages 1374–1382
Alterations to the composition of the bowel microbiota (dysbioses) are associated with particular diseases and conditions of humans. There is a need to discover new, indigestible polysaccharides which are selective growth substrates for commensal bowel bacteria. These substrates (prebiotics) could be added to food in intervention studies to correct bowel dysbiosis. A collection of commensal bacteria was screened for growth in culture using a highly-branched xylan produced by New Zealand flax. Two, Bacteroides ovatus ATCC 8483 and Bacteroides xylanisolvens DSM 18836 grew well on this substrate. The utilisation of the xylan was studied chromatographically and by constituent sugar analysis. The two closely related species utilised the xylan in different ways, and differently from their use of wheat arabinoxylan. The growth of Bacteroides species on other plant xylans having differing chemical structures was also investigated. Novel xylans expand the choice of potential prebiotics that could be used to correct bowel dysbioses.
Dr Manuela Centanni is a Postdoctoral Fellow in Professor Gerald Tannock's lab (which is also affiliated with the research theme Microbiome Otago). Collaborative partners included Victoria University of Wellington's Ferrier Research Institute, Donvis Limited and the Riddet Insitute (both situated in Palmerston North).
M. d. Sainur Samad, Ambarish Biswas, Lars R. Bakken, Timothy J. Clough, Cecile A. M. de Klein, Karl G. Richards, Gary J. Lanigan and Sergio E. Morales
Scientific Reports 2016; 6: 35990. DOI: 10.1038/srep35990
Denitrification is mediated by microbial, and physicochemical, processes leading to nitrogen loss via N2O and N2 emissions. Soil pH regulates the reduction of N2O to N2, however, it can also affect microbial community composition and functional potential. Here we simultaneously test the link between pH, community composition, and the N2O emission ratio (N2O/(NO + N2O + N2)) in 13 temperate pasture soils. Physicochemical analysis, gas kinetics, 16S rRNA amplicon sequencing, metagenomic and quantitative PCR (of denitrifier genes: nirS, nirK, nosZI and nosZII) analysis were carried out to characterize each soil. We found strong evidence linking pH to both N2O emission ratio and community changes. Soil pH was negatively associated with N2O emission ratio, while being positively associated with both community diversity and total denitrification gene (nir & nos) abundance. Abundance of nosZII was positively linked to pH, and negatively linked to N2O emissions. Our results confirm that pH imposes a general selective pressure on the entire community and that this results in changes in emission potential. Our data also support the general model that with increased microbial diversity efficiency increases, demonstrated in this study with lowered N2O emission ratio through more efficient conversion of N2O to N2.
The lead author, Md Sainur Samad, is a PhD student in Sergio Morales' lab along with Postdoctoral Fellow and departmental Bioinformatician Ambarish Biswas. Collaborators are based in Norway and Ireland, and more locally at AgResearch Invermay and the Lincoln University.
Adrian G. Patterson, Simon A. Jackson, Corinda Taylor, Gary B. Evans, George P.C. Salmond, Rita Przybilski, Raymond H.J. Staals, Peter C. Fineran
Molecular Cell, November 2016, DOI: http://dx.doi.org/10.1016/j.molcel.2016.11.012
Bacteria commonly exist in high cell density populations, making them prone to viral predation and horizontal gene transfer (HGT) through transformation and conjugation. To combat these invaders, bacteria possess an arsenal of defenses, such as CRISPR-Cas adaptive immunity. Many bacterial populations coordinate their behavior as cell density increases, using quorum sensing (QS) signaling. In this study, we demonstrate that QS regulation results in increased expression of the type I-E, I-F, and III-A CRISPR-Cas systems in Serratia cells in high-density populations. Strains unable to communicate via QS were less effective at defending against invaders targeted by any of the three CRISPR-Cas systems. Additionally, the acquisition of immunity by the type I-E and I-F systems was impaired in the absence of QS signaling. We propose that bacteria can use chemical communication to modulate the balance between community-level defense requirements in high cell density populations and host fitness costs of basal CRISPR-Cas activity.
The project was led by the Fineran Lab (Department of Microbiology and Immunology, University of Otago) with contributions from Gary Evans (Ferrier Research Institute, Victoria University of Wellington) and George Salmond (Department of Biochemistry, University of Cambridge).
From left to right: Dr Simon Jackson, Dr Raymond Staals, Associate Professor Peter Fineran, Adrian Patterson, Dr Rita Przybilski.
Edward S. Taylor, John L. McCall, Adam Girardin, Fran M. Munro, Michael A. Black and Roslyn A. Kemp
OncoImmunology, Volume 5, 2016 - Issue 11
T cells play a crucial role in preventing the growth and spread of colorectal cancer (CRC). However, immunotherapies against CRC have only shown limited success, which may be due to lack of understanding about the effect of the local tumor microenvironment (TME) on T cell function. The goal of this study was to determine whether T cells in tumor tissue were functionally impaired compared to T cells in non-tumor bowel (NTB) tissue from the same patients. We showed that T cell populations are affected differently by the TME. In the tumor, T cells produced more IL-17 and less IL-2 per cell than their counterparts from NTB tissue. T cells from tumor tissue also had impaired proliferative ability compared to T cells in NTB tissue. This impairment was not related to the frequency of IL-2 producing T cells or regulatory T cells, but T cells from the TME had a higher co-expression of inhibitory receptors than T cells from NTB. Overall, our data indicate that T cells in tumor tissue are functionally altered by the CRC TME, which is likely due to cell intrinsic factors. The TME is therefore an important consideration in predicting the effect of immune modulatory therapies.
Edward Taylor was a PhD student in Dr Roslyn Kemp's lab. The projkect was a collaboration with University of Otago colleagues in the Department of Surgical Sciences and the Department of Biochemistry.
Raymond H. J. Staals, Simon A. Jackson, Ambarish Biswas, Stan J. J. Brouns, Chris M. Brown & Peter C. Fineran
Nature Communications 7, 12853 (2016), doi:10.1038/ncomms12853
Just like humans, bacteria are constantly under attack by viruses that try to infect them by injecting viral DNA. CRISPR-Cas systems can remember and destroy invading DNA by storing short, invader-derived, pieces of DNA (called 'spacers') into their genetic memory banks. A previous study demonstrated that that viruses and plasmids, which can avoid recognition by mutating their DNA, will trigger the bacteria's CRISPR-Cas system to respond by quickly acquiring new immunological memories from these mutated threats. This process is called 'primed adaptation'.
In the current study, the team sought to understand how these new memories were selected during the primed adaptation response. They discovered that the acquisition of memories from plasmids not previously encountered was very inefficient in comparison to the mutated plasmids. By studying the order in which these new memories were acquired, the researchers developed a new model for primed adaptation. The data indicated that acquisition of new immunological memory occurs not only during primed adaptation but also as a consequence of direct defence against invading genetic elements that have not mutated to evade recognition by the CRISPR-Cas system.
CRISPR–Cas systems provide bacteria with adaptive immunity against foreign nucleic acids by acquiring short, invader-derived sequences called spacers. Here, we use high-throughput sequencing to analyse millions of spacer acquisition events in wild-type populations of Pectobacterium atrosepticum. Plasmids not previously encountered, or plasmids that had escaped CRISPR–Cas targeting via point mutation, are used to provoke naive or primed spacer acquisition, respectively. The origin, location and order of spacer acquisition show that spacer selection through priming initiates near the site of CRISPR–Cas recognition (the protospacer), but on the displaced strand, and is consistent with 3′–5′ translocation of the Cas1:Cas2-3 acquisition machinery. Newly acquired spacers determine the location and strand specificity of subsequent spacers and demonstrate that interference-driven spacer acquisition (‘targeted acquisition’) is a major contributor to adaptation in type I-F CRISPR–Cas systems. Finally, we show that acquisition of self-targeting spacers is occurring at a constant rate in wild-type cells and can be triggered by foreign DNA with similarity to the bacterial chromosome.
Michael Petridis, Chelsea Vickers, Jennifer Robson, Joanna L. McKenzie, Magdalena Bereza, Abigail Sharrock, Htin Lin Aung, Vickery L. Arcus and Gregory M. Cook
Journal of Molecular Biology (2016) 428, 4315-4329, http://dx.doi.org/10.1016/j.jmb.2016.09.009
Soil-dwelling bacteria of the phylum actinomycetes generally harbor either GlnR or AmtR as a global regulator of nitrogen metabolism. Mycobacterium smegmatis harbors both of these canonical regulators; GlnR regulates the expression of key genes involved in nitrogen metabolism, while the function and signal transduction pathway of AmtR in M. smegmatis remains largely unknown. Here, we report the structure and function of the M. smegmatis AmtR and describe the role of AmtR in the regulation of nitrogen metabolism in response to nitrogen availability. To determine the function of AmtR in M. smegmatis, we performed genome-wide expression profiling comparing the wild-type versus an ΔamtR mutant and identified significant changes in the expression of 11 genes, including an operon involved in urea degradation. An AmtR consensus-binding motif (CTGTC-N4 -GACAG) was identified in the promoter region of this operon, and ligand-independent, high-affinity AmtR binding was validated by both electrophoretic mobility shift assays and surface plasmon resonance measurements. We confirmed the transcription of a cis -encoded small RNA complementary to the gene encoding AmtR under nitrogen excess, and we propose a post-transcriptional regulatory mechanism for AmtR. The three-dimensional X-ray structure of AmtR at 2.0 Å revealed an overall TetR-like dimeric structure, and the alignment of the M. smegmatis AmtR and Corynebacterium glutamicum AmtR regulatory domains showed poor structural conservation, providing a potential explanation for the lack of M. smegmatis AmtR interaction with the adenylylated PII protein. Taken together, our data suggest an AmtR (repressor)/GlnR (activator) competitive binding mechanism for transcriptional regulation of urea metabolism that is controlled by a cis -encoded small antisense RNA.
The project was a collaboration between the University of Otago's Department of Microbiology and Immunology, the University of Waikato's Department of Biological Sciences and the Maurice Wilkins Centre for Molecular Biodiversity, with funding provided by the Maurice Wilkins Centre and a Marsden Grant from the Royal Society of New Zealand.
Michael Petridis, now a Postdoctoral Fellow in the Cook Lab, undertook this work as a part of a PhD with the Department of Microbiology and Immunology. He was supported by a University of Otago Doctoral Scholarship, the Webster Center for Infectious Diseases and the Otago School of Medical Sciences.
Artur Muszyński, Christian Heiss, Christian T. Hjuler, John T. Sullivan, Simon J. Kelly, Mikkel B. Thygesen, Jens Stougaard, Parastoo Azadi, Russell W. Carlson, and Clive W. Ronson
The Journal of Biological Chemistry, August 2016
In the symbiosis formed between Mesorhizobium loti strain R7A and Lotus japonicus Gifu, rhizobial exopolysaccharide (EPS) plays an important role in infection thread formation. Mutants of strain R7A affected in early exopolysaccharide biosynthetic steps form nitrogen-fixing nodules on L. japonicus Gifu after a delay, whereas mutants affected in mid or late biosynthetic steps induce uninfected nodule primordia. Our recent collaborative studies (Kawaharada, Y. et al. (2015) Nature 523, 308–312) showed that M. loti low molecular mass exopolysaccharides are perceived by the L. japonicus receptor-like kinase, EPR3, and serve as signal molecules that regulate infection thread formation and bacterial passage through the plant’s epidermal cell layer during root nodule formation. In the current paper, we characterize in detail the structures of high and low molecular mass EPS isolated from M. loti R7A including the locations of non-stoichiometric O-acetyl substituents. We demonstrate that the repeating unit of EPS polymer consists of highly O-acetylated octasaccharide with acidic branch substituted with an a-d-glucuronic and β-d-riburonic acids. In addition we propose that the low molecular mass octasaccharide EPS that is recognized by EPR3 kinase receptor is the monomer of the biosynthetic repeating oligosaccharide unit of the high molecular mass polymeric EPS. The availability of these structures will facilitate studies of EPR3 receptor binding of symbiotically compatible and incompatible EPS and lead to a better understanding of the positive or negative consequences on infection by the M. loti exo mutants synthesizing such EPS variants.
This paper is the outcome of a truly global collaboration involving researchers in four institutions and three countries, the United States, Denmark and New Zealand. The work was carried out under the umbrella of the Centre for Carbohydrate Recognition and Signalling (carb.au.dk), a Centre of Excellence supported financially by the Danish National Research Foundation and led by Professor Jens Stougaard at the University of Aarhus. The work was led by Professor Clive Ronson from the University of Otago who provided the biological expertise, and by Dr Artur Muszyński at the Centre for Complex Carbohydrate Research in Athens, Georgia, USA, who led the chemistry work, and they are joint corresponding authors on the paper. Researchers at the University of Copenhagen also made important contributions to the chemical characterization, especially of the low molecular mass EPS. Other contributors from Otago were Simon Kelly who was a PhD student for most of the study and who is currently a postdoctoral fellow at Aarhus, and Dr John Sullivan who is an assistant research fellow in the Ronson lab.
The authors also supplied the journal cover image. Photo credit: Stefan Eberhard, Christian T. Hjuler, and Artur Muszyński.
SEM (×370) image of freeze-dried extract of exopolysaccharide (EPS) isolated from Mesorhizobium loti strain R7A. This high molecular mass polymer consists of O-acetylated octasaccharide repeating units. A single monomeric O-acetylated octasaccharide, demonstrated in the lowest energy 3D structural model (overlay), is a biosynthetic repeat of EPS and plays a signaling role in receptor-mediated symbiotic infection of Lotus japonicus by M. loti.
Colin Davies, Vernon K. Ward
PLOS One, August 2016
Murine norovirus-1 (MNV-1) is known to subvert host cell division inducing an accumulation of cells in the G0/G1 phase, creating conditions where viral replication is favored. This study identified that NS5 (VPg), is capable of inducing cell cycle arrest in the absence of viral replication or other viral proteins in an analogous manner to MNV-1 infection. NS5 expression induced an accumulation of cells in the G0/G1 phase in an asynchronous population by inhibiting progression at the G1/S restriction point. Furthermore, NS5 expression resulted in a down-regulation of cyclin A expression in asynchronous cells and inhibited cyclin A expression in cells progressing from G1 to S phase. The activity of NS5 on the host cell cycle occurs through an uncharacterized function. Amino acid substitutions of NS5(Y26A) and NS5(F123A) that inhibit the ability for NS5 to attach to RNA and recruit host eukaryotic translation initiation factors, respectively, retained the ability to induce an accumulation of cells in the G0/G1 phase as identified for wild-type NS5. To the best of our knowledge, this is the first report of a VPg protein manipulating the host cell cycle.
Colin Davies recently completed his PhD in the Department of Microbiology, supervised by Professor Vernon Ward.
Morad-Rémy Muhsin-Sharafaldine, Sarah C. Saunderson, Amy C. Dunn, James M. Faed, Torsten Kleffmann, Alexander D. McLellan
Oncotarget, June 2016
The study shows that tiny vesicles released from the surface of tumour cells during chemotherapy contribute to thrombotic events in cancer patients. These disorders of the coagulation system, such as deep vein thrombosis and pulmonary embolism, are the second highest cause of death in cancer patients. The work carried out by lead author Remy Muhsin (PhD student) has identified a new system of pathological coagulation that is a rapid, short circuit version of physiological coagulation
Extracellular vesicles (EV) are lipid particles released from eukaryotic cells into the extracellular fluid. Depending on the cell type or mechanism of release, vesicles vary in form and function and exert distinct functions in coagulation and immunity. Tumor cells may constitutively shed vesicles known as exosomes or microvesicles (MV). Alternatively, apoptosis induces the release of apoptotic blebs or vesicles (ApoV) from the plasma membrane. EV have been implicated in thrombotic events (the second highest cause of death in cancer patients) and tumor vesicles contribute to the anti-cancer immune response. In this study, we utilized the well characterized B16 melanoma model to determine the molecular composition and procoagulant and immunogenic potential of exosomes, MV and ApoV. Distinct patterns of surface and cytoplasmic molecules (tetraspanins, integrins, heat shock proteins and histones) were expressed between the vesicle types. Moreover, in vitro coagulation assays revealed that membrane-derived vesicles, namely MV and ApoV, were more procoagulant than exosomes–with tissue factor and phosphatidylserine critical for procoagulant activity. Mice immunized with antigen-pulsed ApoV and challenged with B16 tumors were protected out to 60 days, while lower protection rates were afforded by MV and exosomes. Together the results demonstrate distinct phenotypic and functional differences between vesicle types, with important procoagulant and immunogenic functions emerging for membrane-derived MV and ApoV versus endosome-derived exosomes. This study highlights the potential of EV to contribute to the prothrombotic state, as well as to anti-cancer immunity.
April Pawluk, Raymond H.J. Staals, Corinda Taylor, Bridget N.J. Watson, Senjuti Saha, Peter C. Fineran, Karen L. Maxwell and Alan R. Davidson
Nature Microbiology, vol 1, August 2016
The authors of this paper have uncovered a new arsenal of anti-CRISPR proteins produced by viruses that enable them to circumvent CRISPR-Cas adaptive immunity in their arms race with bacteria. CRISPR-Cas systems come in many different varieties, and remarkably, one of the newly discovered anti-CRISPR proteins was dual specificity, inhibiting the activity of two types of CRISPR-Cas systems. The apparent widespread distribution of anti-CRISPRs suggest that phages and plasmids can evade bacterial immunity to allow their transfer between bacteria.
CRISPR-Cas systems provide sequence-specific adaptive immunity against foreign nucleic acids. They are present in approximately half of all sequenced prokaryotes3 and are expected to constitute a major barrier to horizontal gene transfer. We previously described nine distinct families of proteins encoded in Pseudomonas phage genomes that inhibit CRISPR-Cas function. We have developed a bioinformatic approach that enabled us to discover additional anti-CRISPR proteins encoded in phages and other mobile genetic elements of diverse bacterial species. We show that five previously undiscovered families of anti-CRISPRs inhibit the type I-F CRISPR-Cas systems of both Pseudomonas aeruginosa and Pectobacterium atrosepticum, and a dual specificity anti-CRISPR inactivates both type I-F and I-E CRISPR-Cas systems. Mirroring the distribution of the CRISPR-Cas systems they inactivate, these anti-CRISPRs were found in species distributed broadly across the phylum Proteobacteria. Importantly, anti-CRISPRs originating from species with divergent type I-F CRISPR-Cas systems were able to inhibit the two systems we tested, highlighting their broad specificity. These results suggest that all type I-F CRISPR-Cas systems are vulnerable to inhibition by anti- CRISPRs. Given the widespread occurrence and promiscuous activity of the anti-CRISPRs described here, we propose that anti-CRISPRs play an influential role in facilitating the movement of DNA between prokaryotes by breaching the barrier imposed by CRISPR-Cas systems.
The people involved
The project was led from the groups of Alan Davidson and Karen Maxwell from the University of Toronto. Collaborators from Associate Professor Peter Fineran’s group were Postdoctoral Fellow Dr Raymond Staals, Research Assistant Corinda Taylor and PhD student Bridget Watson.
Prashanth Thevkar Nagesh, Matloob Husain
Journal of Virology, 90(9) May 2016
Influenza A virus (IAV) continues to significantly impact global public health by causing regular seasonal epidemics, occasional pandemics, and zoonotic outbreaks. IAV is among the successful human viral pathogens that has evolved various strategies to evade host defenses, prevent the development of a universal vaccine, and acquire antiviral drug resistance. A comprehensive knowledge of IAV-host interactions is needed to develop a novel and alternative anti-IAV strategy. Host produces a variety of factors that are able to fight IAV infection by employing various mechanisms. However, the full repertoire of anti-IAV host factors and their antiviral mechanisms has yet to be identified. We have identified here a new host factor, histone deacetylase 1 (HDAC1) that inhibits IAV infection. We demonstrate that HDAC1 is a component of host innate antiviral response against IAV, and IAV undermines HDAC1 to limit its role in antiviral response.
Viruses dysregulate the host factors that inhibit virus infection. Here, we demonstrate that human enzyme, histone deacetylase 1 (HDAC1) is a new class of host factor that inhibits influenza A virus (IAV) infection, and IAV dysregulates HDAC1 to efficiently replicate in epithelial cells. A time-dependent decrease in HDAC1 polypeptide level was observed in IAV-infected cells, reducing to<50% by 24 h of infection. A further depletion (97%) of HDAC1 expression by RNA interference increased the IAV growth kinetics, increasing it by>3-fold by 24 h and by>6-fold by 48 h of infection. Conversely, overexpression of HDAC1 decreased the IAV infection by>2-fold. Likewise, a time-dependent decrease in HDAC1 activity, albeit with slightly different kinetics to HDAC1 polypeptide reduction, was observed in infected cells. Nevertheless, a further inhibition of deacetylase activity increased IAV infection in a dose-dependent manner. HDAC1 is an important host deacetylase and, in addition to its role as a transcription repressor, HDAC1 has been lately described as a coactivator of type I interferon response. Consistent with this property, we found that inhibition of deacetylase activity either decreased or abolished the phosphorylation of signal transducer and activator of transcription I (STAT1) and expression of interferon-stimulated genes, IFITM3, ISG15, and viperin in IAV-infected cells. Furthermore, the knockdown of HDAC1 expression in infected cells decreased viperin expression by 58% and, conversely, the overexpression of HDAC1 increased it by 55%, indicating that HDAC1 is a component of IAV-induced host type I interferon antiviral response.
The people involved
This paper is a direct output from PhD thesis research by Prashanth Thevkar Nagesh (Department of Microbiology and Immunology). Dr Matloob Husain is his supervisor.
Samuel E Norton, Elliott TJ Dunn, John L McCall, Fran Munro and Roslyn A Kemp
Clinical & Translational Immunology (2016) 5, e76; doi:10.1038/cti.2016.21
In contrast to many cancers, a high infiltration of macrophages in colorectal cancer (CRC) has been associated with improved prognosis for patients. Cytokines and other stimuli from the tumor microenvironment affect monocyte to macrophage maturation and subsequent phenotype and function. Heterogeneous myeloid populations were identified using a novel flow cytometry panel in both tumor and paired non-tumor bowel (NTB) from CRC patients. The frequency of macrophage subsets with a gut-conditioned phenotype was lower in tumor compared with NTB. We used an in vitro system to show that two of the macrophage populations represented pro-inflammatory and anti-inflammatory phenotypes. Conditioned media that contained high levels of interleukin-6 promoted and maintained an anti-inflammatory phenotype in vitro. This study demonstrates the plasticity and heterogeneity of macrophage subtypes in human CRC, and the feasibility of studying complex populations. Ex vivo experiments demonstrate that macrophage subsets are influenced by the tumor microenvironment.
The people involved
This work was undertaken as part of Sam Norton's PhD research. Dr Roslyn Kemp, Department of Microbiology and Immunology is his supervisor, and Elliott Dunn is a former student of the Kemp Lab. John McCall and Fran Munro are based in the University of Otago's Department of Surgical Sciences.
Tania L. Slatter, Michelle Wilson, Chingwen Tang, Hamish G. Campbell, Vernon K. Ward, Vivienne L. Young, David Van Ly, Nicholas I. Fleming, Antony W. Braithwaite & Margaret A. Baird
OncoImmunology, 5(3), e1112941. doi: 10.1080/2162402X.2015.1112941
Summary of the work
In this project it was found that the tumour supressor protein p53 plays an important role in helping APC (activated antigen-presenting cells) generate an antitumour response. This demonstrated a completely new role for this protein and could contribute to future immunotherapies.
Activated antigen-presenting cells (APC) deliver the three signals cytotoxic T cells require to differentiate into effector cells that destroy the tumor. These comprise antigen, co-stimulatory signals and cytokines. Once these cells have carried out their function, they apoptose. We hypothesized that the tumor suppressor protein, p53, played an important role in generating the antitumor response facilitated by APC. CD11cC APC derived from p53 wild-type (wt) mouse (wt p53) GM-CSF bone marrow cultures (BMAPC) and activated had reduced survival compared to BMAPC from p53 null consistent with p53-mediated apoptosis following activation. There was a lower percentage of antigenic peptide/MHC I complexes on antigen-pulsed p53 null cells suggesting p53 played a role in antigen processing but there was no difference in antigen-specific T cell proliferative responses to these cells in vivo. In contrast, antigenspecific cytotoxicity in vivo was markedly reduced in response to p53 null BMAPC. When these cells were pulsed with a model tumor antigen and delivered as a prophylactic vaccination, they provided no protection against melanoma cell growth whereas wt BMAPC were very effective. This suggested that p53 might regulate the requisite third signal and, indeed, we found that p53 null BMAPC produced less IL-12 than wt p53 BMAPC and that p53 bound to the promoter region of IL-12. This work suggests that p53 in activated BMAPC is associated with the generation of IL-12 required for the differentiation of cytotoxic immune responses and an effective antitumor response. This is a completely new role for this protein that has implications for BMAPC-mediated immunotherapy.
The people involved
Michelle Wilson is the Department of Microbiology and Immunology's Flow Cytometry Technician and Professor Vernon Ward is a virology researcher. This work was undertake alongside colleagues from the Department of Pathology, the Maurice Wilkins Centre and the University of Sydney.
Linda M. Samuelsson, Wayne Young, Karl Fraser, Gerald W. Tannock, Julian Lee, Nicole C. Roy
Metabolomics (March 2016) DOI 10.1007/s11306-016-1016-7
Introduction: Digestion resistant carbohydrates (DRC) are complex carbohydrates that resist digestion and absorption in the small bowel. Diets high in DRC can have wide ranging impacts on the health of the host, which include changes to immunity and allergy, incidence of cardiovascular disease, and obesity.
Objectives: The aim of this study was to characterise the effects of DRC (inulin, konjac or resistant starch) on large intestinal short-chain fatty acid (SCFA) concentrations and serum metabolite and lipid profiles.
Methods: A rat model was used to compare the effects of feeding a basal diet or the basal diet containing 5% inulin, konjac or resistant starch for 14 days.
Results: Of the three DRC, inulin had the greatest effect; ten serum phospholipids differed significantly in abundance between inulin-treated and control rats. In particular phosphatidylcholines and lysophosphatidylcholines containing fatty acyl chains 22:5, 22:4, 20:4, 18:0 and 16:0 were increased in the inulin-fed group, whereas phosphocholines containing fatty acyls 20:5 and 22:6 were decreased.
Conclusion: These results indicated an impact on both n -3 and n -6 fatty acid metabolism as a result of inulin dietary intake. Increased intestinal concentrations of SCFA were detected in rats fed DRC, but only inulin caused appreciable changes to serum lipid profiles.
The people involved
Professor Gerald Tannock is a Principal Investigator in the Department of Microbiology and Immunology. This work was undertaken with other New Zealand researchers from AgResearch, the Riddet Centre of Research Excellence, Plant and Food Research and Gravida National Centre for Growth and Development.
Moran Yassour, Mi Young Lim, Hyun Sun Yun, Timothy L. Tickle, Joohon Sung, Yun-Mi Song, Kayoung Lee, Eric A. Franzosa, Xochitl C. Morgan, Dirk Gevers, Eric S. Lander, Ramnik J. Xavier, Bruce W. Birren, GwangPyo Ko and Curtis Huttenhower
Genome Medicine (February 2016) DOI 10.1186/s13073-016-0271-6
Background: Obesity and type 2 diabetes (T2D) are linked both with host genetics and with environmental factors, including dysbioses of the gut microbiota. However, it is unclear whether these microbial changes precede disease onset. Twin cohorts present a unique genetically-controlled opportunity to study the relationships between lifestyle factors and the microbiome. In particular, we hypothesized that family-independent changes in microbial composition and metabolic function during the sub-clinical state of T2D could be either causal or early biomarkers of progression.
Methods: We collected fecal samples and clinical metadata from 20 monozygotic Korean twins at up to two time points, resulting in 36 stool shotgun metagenomes. While the participants were neither obese nor diabetic, they spanned the entire range of healthy to near-clinical values and thus enabled the study of microbial associations during sub-clinical disease while accounting for genetic background.
Results: We found changes both in composition and in function of the sub-clinical gut microbiome, including a decrease in Akkermansia muciniphila suggesting a role prior to the onset of disease, and functional changes reflecting a response to oxidative stress comparable to that previously observed in chronic T2D and inflammatory bowel diseases. Finally, our unique study design allowed us to examine the strain similarity between twins, and we found that twins demonstrate strain-level differences in composition despite species-level similarities.
Conclusions: These changes in the microbiome might be used for the early diagnosis of an inflamed gut and T2D prior to clinical onset of the disease and will help to advance toward microbial interventions.
Noelyn A. Hung, Ramona A. Eiholzer, Stenar Kirs, Jean Zhou, Kirsten Ward-Hartstonge, Anna K. Wiles, Chris M. Frampton, Ahmad Taha, Janice A. Royds and Tania L. Slatter
Modern Pathology advance online publication, 15 January 2016; doi:10.1038/modpathol.2015.156
Summary of the work
Using RNA-Seq and immunohistochemistry microscopy, it was determined that a majority of patients with no defined telomere-maintenance mechanism contained high infiltrates of tumour-associated macrophages in Glioblastoma. Patients with a high infiltrate of tumour-associated macrophages were associated with poorer patient outcomes, suggesting that these may be a potential target for treatment in glioblastoma.
Telomere maintenance is a hallmark of cancer and likely to be targeted in future treatments. In glioblastoma established methods of identifying telomerase and alternative lengthening of telomeres leave a significant proportion of tumors with no defined telomere maintenance mechanism. This study investigated the composition of these tumors using RNA-Seq. Glioblastomas with an indeterminate telomere maintenance mechanism had an increased immune signature compared with alternative lengthening of telomeres and telomerase-positive tumors. Immunohistochemistry for CD163 confirmed that the majority (80%) of tumors with an indeterminate telomere maintenance mechanism had a high presence of tumor-associated macrophages. The RNA-Seq and immunostaining data separated tumors with no defined telomere maintenance mechanism into three subgroups: alternative lengthening of telomeres like tumors with a high presence of tumor-associated macrophages and telomerase like tumors with a high presence of tumor-associated macrophages. The third subgroup had no increase in tumor-associated macrophages and may represent a distinct category. The presence of tumorassociated macrophages conferred a worse prognosis with reduced patient survival times (alternative lengthening of telomeres with and without macrophages P = 0.0004, and telomerase with and without macrophages P = 0.013). The immune signatures obtained from RNA-Seq were significantly different between telomere maintenance mechanisms. Alternative lengthening of telomeres like tumors with macrophages had increased expression of interferon-induced proteins with tetratricopeptide repeats (IFIT1–3). Telomerase-positive tumors with macrophages had increased expression of macrophage receptor with collagenous structure (MARCO), CXCL12 and sushi-repeat containing protein x-linked 2 (SRPX2). Telomerase-positive tumors with macrophages were also associated with a reduced frequency of total/near total resections (44% vs 476% for all other subtypes, P = 0.014). In summary, different immune signatures are found among telomere maintenance mechanism-based subgroups in glioblastoma. The reduced extent of surgical resection of telomerase-positive tumors with macrophages suggests that some tumor-associated macrophages are more unfavorable.
The people involved
Kirsten Ward Hartstonge was funded by The Centre for Translational Cancer to complete a Summer Studentship (2013/2014). The project was a collaboration between Dr Roslyn Kemp (Department of Microbiology and Immunology) and Dr Tania Slatter (Department of Pathology). The Gut Health Network funded the consumables.
Dunn, E.T.J., Taylor, E.S., Stebbings, S., Schultz, M., Butt, A.G. and Kemp, R.A.
Immunology and Cell Biology (2015), 1-9. doi:10.1038/icb.2015.112.
Crohn's disease (CD) is an inflammatory bowel disease characterized by patchy inflammation of the gastrointestinal tract. Ankylosing spondylitis (AS) is primarily characterized by inflammation of the lower vertebral column, and many patients with AS present with inflammatory gut symptoms. Genome wide association studies have highlighted significant overlap in short nucleotide polymorphisms for both diseases. We hypothesized that patients with CD and AS have a common intestinal immune signature, characterized by inflammatory T cells, compared with healthy people. We designed a pilot study to determine both the feasibility of defining complex immune signatures from primary tissue, and differences in the local immune signature of people with inflammatory diseases compared with healthy people. Intestinal biopsies were obtained by colonoscopy from healthy patients, non-inflamed regions of CD patients and AS patients with inflammatory gut symptoms. A flow cytometry platform was developed measuring polyfunctional T cell populations based on cytokines, surface molecules and transcription factors. There was overlap in the immune signature of people with CD or AS, characterized by changes in the frequency of regulatory T cells, compared with healthy people. There were significant differences in frequencies of other polyfunctional T cell populations-CD patients had an increased frequency of T cells producing IL-22 and IFNγ, while AS patients had an increased frequency of T cells producing IL-2; compared with healthy people. These data indicate that the local immune signature could be described in these patients and that distinct immune mechanisms may underlie disease progression.
The people involved
Elliott Dunn is a MSc graduate of Ros Kemp's lab, and Edward Taylor is a current PhD student. Simon Stebbings and Michael Schultz are based in the University of Otago's Department of Medicine, and Grant Butt is in the Department of Physiology.
Vivek Poonthiyil, Prashanth T. Nagesh, Matloob Husain, Vladimir B. Golovko and Antony J. Fairbanks
ChemistryOpen, December 2015, DOI: 10.1002/open.201500109
Summary of the work
This paper describes the discovery that carbohydrate molecules isolated from a cheap source – chicken eggs – and immobilized on gold nanoparticles can be used for the selective detection of influenza virus in a sample. Therefore, this finding has the potential for developing a cheaper diagnostic method for detecting influenza virus.
The associated artwork was chosen for the cover of this issue of ChemistryOpen. The cover picture shows the isolation of complex bi-antennary oligosaccharides from hens’ eggs and their conjugation to gold nanoparticles. Gold nanoparticles carrying these sugars can then bind to specific receptors (hemagglutinin) on the surface of the influenza virus, causing particle aggregation, which changes their spectroscopic properties. Upon aggregation, they undergo a red-shift in their surface plasmon resonance, as illustrated by the bound particles shining in the cover image. These changes in spectroscopic properties are the basis of a detection system capable of detecting viral hemagglutinin at nanomolar concentrations, as well as the virus itself.
Gold nanoparticles decorated with full-length sialic acid terminated complex bi-antennary N-glycans, synthesized with glycans isolated from egg yolk, were used as a sensor for the detection of both recombinant hemagglutinin (HA) and whole influenza A virus particles of the H1N1 subtype. Nanoparticle aggregation was induced by interaction between the sialic acid termini of the glycans attached to gold and the multivalent sialic acid binding sites of HA. Both dynamic light scattering (DLS) and UV/Vis spectroscopy demonstrated the efficiency of the sensor, which could detect viral HA at nanomolar concentrations and revealed a linear relationship between the extent of nanoparticle aggregation and the concentration of HA. UV/Vis studies also showed that these nanoparticles can selectively detect an influenza A virus strain that preferentially binds sialic acid terminated glycans with a(2!6) linkages over a strain that prefers glycans with terminal a(2!3)-linked sialic acids.
Ryan Harvey, Catherine McCaughan, Lyn M. Wise, Andrew A. Mercer, Stephen B. Fleming
Virus Research 208 (2015) 180–188
Summary of the work
Interferons (IFNs) are a multifunctional family of cytokines that play a critical role as a first line of defence against viral infection. Activation of the Janus Kinase/signal transducer and activation of transcription (JAK/STAT) signalling pathway by IFNs leads to the production of numerous IFN stimulated genes (ISGs) that block viral replication. Our findings show that the parapoxvirus, orf virus blocks the expression of ISGs induced by type I and type II IFNs. This is the first report that describes the inhibition of ISG expression by a parapoxvirus. Moreover we have shown that Orf virus has evolved a mechanism to impair the JAK/STAT signalling pathway by dephosphorylating STAT1 in virus-infected cells that does not require new viral gene synthesis.
Interferons (IFNs) play a critical role as a first line of defence against viral infection. Activation of the Janus kinase/signal transducer and activation of transcription (JAK/STAT) pathway by IFNs leads to the production of IFN stimulated genes (ISGs) that block viral replication. The Parapoxvirus, Orf virus (ORFV) induces acute pustular skin lesions of sheep and goats and is transmissible to man. The virus replicates in keratinocytes that are the immune sentinels of skin. We investigated whether or not ORFV could block the expression of ISGs. The human gene GBP1 is stimulated exclusively by type II IFN while MxA is stimulated exclusively in response to type I IFNs. We found that GBP1 and MxA were strongly inhibited in ORFV infected HeLa cells stimulated with IFN-γ or IFN-α respectively. Furthermore we showed that ORFV inhibition of ISG expression was not affected by cells pretreated with adenosine N1-oxide (ANO), a molecule that inhibits poxvirus mRNA translation. This suggested that new viral gene synthesis was not required and that a virion structural protein was involved. We next investigated whether ORFV infection affected STAT1 phosphorylation in IFN-γ or IFN-α treated HeLa cells. We found that ORFV reduced the levels of phosphorylated STAT1 in a dose-dependent manner and was specific for Tyr701 but not Ser727. Treatment of cells with sodium vanadate suggested that a tyrosine phosphatase was responsible for dephosphorylating STAT1-p. ORFV encodes a factor, ORFV057, with homology to the vaccinia virus structural protein VH1 that impairs the JAK/STAT pathway by dephosphorylating STAT1. Our findings show that ORFV has the capability to block ISG expression and modulate the JAK/STAT signalling pathway.
The people involved
Ryan Harvey completed an MSc under the supervision of Dr Steve Fleming in the Virus Research Unit (VRU), Department of Microbiology and Immunology, in 2014. Catherine McCaughan was a technician in the VRU and retired earlier this year. Dr Lyn Wise is a Research Fellow in the VRU and Professor Andy Mercer is the Director of the VRU.
Adrian G. Patterson, James T. Chang, Corinda Taylor and Peter C. Fineran
Nucleic Acids Research, 2015, doi: 10.1093/nar/gkv517
Summary of the work
In this paper, the authors show that the expression of the Type I-F CRISPR-Cas system is controlled by CRP-cAMP and GalM. Mutation of the genes encoding these cellular components influences CRISPR-Cas acquisition and interference processes. These findings suggest that a metabolite-sensing regulatory network exists and that perturbation of normal metabolic flux within the cell may trigger CRISPR-Cas activity.
The people involved
Adrian Patterson is a second-year PhD student under the supervision of Associate Professor Peter Fineran. James Chang is a former Msc student of Peter's who is carrying out his PhD under the supervision of Professor Clive Ronson. Corinda Taylor works as a Research Technician in the Fineran Lab.
Chris Greening, Carlo R. Carere, Rowena Rushton-Green, Liam K. Harold, Kiel Hards, Matthew C. Taylor, Sergio E. Morales, Matthew B. Stott, and Gregory M. Cook
PNAS, August 18, 2015, vol. 112 no. 33
Summary of the work
The mechanisms that the “dormant microbial majority” use to remain energized in nutrient-starved soil ecosystems have long remained elusive. In this work, we used an isolate of the highly abundant but poorly understood soil phylum Acidobacteria as a model for understanding microbial persistence mechanisms. When the bacterium entered a persistent state due to nutrient-exhaustion, we showed it could scavenge the trace concentrations of molecular hydrogen gas (H2) found in ambient air using a specialized high-affinity enzyme. These findings demonstrate that Acidobacteria can consume H2 and contribute to global hydrogen cycling. We propose that consumption of trace gases such as H2 provides a dependable general mechanism for dominant soil phyla to generate the maintenance energy required for long-term survival.
The people involved
Lead author Chris Greening is a PhD graduate of the Department of Microbiology and Immunology, while Liam Harold and Kiel Hards are current PhD students and Rowena Rushton-Green is an undergraduate student. Sergio Morales and Greg Cook are academic researchers in the department. The work was undertaken with colleagues from CSIRO (Australia) and GNS Science (New Zealand).
Y. Kawaharada, S. Kelly, M. Wibroe Nielsen, C. T. Hjuler, K. Gysel, A. Muszyński, R. W. Carlson, M. B. Thygesen, N. Sandal, M. H. Asmussen, M. Vinther, S. U. Andersen, L. Krusell, S. Thirup, K. J. Jensen, C. W. Ronson, M. Blaise, S. Radutoiu and J. Stougaard
Nature 523, 308–312 (16 July 2015) doi:10.1038/nature14611
Summary of the work
Clover and other legumes form a unique symbiotic relationship with bacteria known as rhizobia, which they allow to infect their roots. This leads to root nodules being formed in which the bacteria convert nitrogen from the air into ammonia that the plant can use for growth. This study elucidates how legumes perceive and distinguish compatible bacteria based on the exopolysaccharides featuring on the invading cells’ surfaces. The findings have implications for improving the understanding of how other plant and animal species interact with bacteria in their environment and defend themselves against hostile infections.
Surface polysaccharides are important for bacterial interactions with multicellular organisms, and some are virulence factors in pathogens. In the legume–rhizobium symbiosis, bacterial exopolysaccharides (EPS) are essential for the development of infected root nodules. We have identified a gene in Lotus japonicus, Epr3, encoding a receptor-like kinase that controls this infection. We show that epr3 mutants are defective in perception of purified EPS, and that EPR3 binds EPS directly and distinguishes compatible and incompatible EPS in bacterial competition studies. Expression of Epr3 in epidermal cells within the susceptible root zone shows that the protein is involved in bacterial entry, while rhizobial and plant mutant studies suggest that Epr3regulates bacterial passage through the plant’s epidermal cell layer. Finally, we show that Epr3expression is inducible and dependent on host perception of bacterial nodulation (Nod) factors. Plant–bacterial compatibility and bacterial access to legume roots is thus regulated by a two-stage mechanism involving sequential receptor-mediated recognition of Nod factor and EPS signals.
The people involved
This research was the result of a collaboration between the Department of Microbiology and Immunology's Professor Clive Ronson and colleagues in Denmark at the Centre for Carbohydrate Recognition and Signalling (CARB).
Maggie Chan, Heather J. L. Brooks, Stephen C. Moratti, Lyall R. Hanton and Jaydee D. Cabral
International Journal of Molecular Sciences, 16 June 2015
Summary of the work
The results of this study demonstrate the importance of oxidation level in the biocompatibility and antimicrobial efficacy of a surgical hydrogel. Although the hydrogel tested offers reduced antimicrobial ability when compared to the higher dextran aldehyde alternative, it functioned effectively as a physical adhesion barrier when used with dressings. A more moderate level of oxidation or the inclusion of broad-spectrum antimicrobial agents could be investigated in future to combine the benefits of biocompatibility, antimicrobial efficacy and adhesion prevention. This could lead to reduced healing times, lowered infection rates, and eliminate the need for second look surgeries to remove adhesions.
A highly oxidized form of a chitosan/dextran-based hydrogel (CD-100) containing 80% oxidized dextran aldehyde (DA-100) was developed as a post-operative aid, and found to significantly prevent adhesion formation in endoscopic sinus surgery (ESS). However, the CD-100 hydrogel showed moderate in vitro cytotoxicity to mammalian cell lines, with the DA-100 found to be the cytotoxic component. In order to extend the use of the hydrogel to abdominal surgeries, reformulation using a lower oxidized DA (DA-25) was pursued. The aim of the present study was to compare the antimicrobial efficacy, in vitro biocompatibility and wound healing capacity of the highly oxidized CD-100 hydrogel with the CD-25 hydrogel. Antimicrobial studies were performed against a range of clinically relevant abdominal microorganisms using the micro-broth dilution method. Biocompatibility testing using human dermal fibroblasts was assessed via a tetrazolium reduction assay (MTT) and a wound healing model. In contrast to the original DA-100 formulation, DA-25 was found to be non-cytotoxic, and showed no overall impairment of cell migration, with wound closure occurring at 72 h. However, the lower oxidation level negatively affected the antimicrobial efficacy of the hydrogel (CD-25). Although the CD-25 hydrogel’s antimicrobial efficacy and anti-fibroblast activity is decreased when compared to the original CD-100 hydrogel formulation, previous in vivo studies show that the CD-25 hydrogel remains an effective, biocompatible barrier agent in the prevention of postoperative adhesions.
The people involved
Maggie Chan is a graduate and former Research Assistant, who worked under the supervision of Dr Heather Brooks from the Department of Microbiology and Immunology. Stephen Moratti, Lyall Hanton and Jadee Cabral are project collaborators from the Department of Chemistry.
Elaine Chiu, Marcel Hijnen, Richard D. Bunker, Marion Boudes, Chitra Rajendran, Kaheina Aizel, Vincent Oliéric, Clemens Schulze-Briese, Wataru Mitsuhashi, Vivienne Young, Vernon K. Ward, Max Bergoin, Peter Metcalf, and Fasséli Coulibaly
PNAS, March 31, 2015
Summary of the work
In this paper, the authors present the structure of protein crystals that form naturally in virally infected insects and boost the insecticidal activity of oral pathogens. These findings may guide their use as synergetic additives to common bioinsecticides. X-ray crystallography is a powerful approach for understanding the structure and function of biological macromolecules, but has so far been largely limited to molecules that form high-quality crystals in the laboratory.
The great benefits that chemical pesticides have brought to agriculture are partly offset by widespread environmental damage to nontarget species and threats to human health. Microbial bioinsecticides are considered safe and highly specific alternatives but generally lack potency. Spindles produced by insect poxviruses are crystals of the fusolin protein that considerably boost not only the virulence of these viruses but also, in cofeeding experiments, the insecticidal activity of unrelated pathogens. However, the mechanisms by which spindles assemble into ultra-stable crystals and enhance virulence are unknown. Here we describe the structure of viral spindles determined by X-ray microcrystallography from in vivo crystals purified from infected insects. We found that a C-terminal molecular arm of fusolin mediates the assembly of a globular domain, which has the hallmarks of lytic polysaccharide monooxygenases of chitinovorous bacteria. Explaining their unique stability, a 3D network of disulfide bonds between fusolin dimers covalently crosslinks the entire crystalline matrix of spindles. However, upon ingestion by a new host, removal of the molecular arm abolishes this stabilizing network leading to the dissolution of spindles. The released monooxygenase domain is then free to disrupt the chitin-rich peritrophic matrix that protects insects against oral infections. The mode of action revealed here may guide the design of potent spindles as synergetic additives to bioinsecticides.
The people involved
Vivienne Young and Professor Ward are based in the Department of Microbiology and Immunology at the University of Otago. Colleagues Elaine Chiu, Richard Bunker and Peter Metcalf are with Auckland University's School of Biological Sciences, with other authors of the paper based in Australia, Switzerland, Japan and France.
David W. Taylor, Yifan Zhu, Raymond H. J. Staals, Jack E. Kornfeld, Akeo Shinkai, John van der Oost, Eva Nogales, Jennifer A. Doudna
Science, April 2015.
In the paper, the authors show how CRISPR-Cas, a surveillance complex in the bacterial immune system, is able to target specific sites on RNA molecules to destroy invading viruses and other foreign genetic elements. The finding could lead to the development of tailor-made RNA-editing tools.
Adaptive immunity in bacteria involves RNA-guided surveillance complexes that use CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target invasive nucleic acids for degradation. While Type I and Type II CRISPR-Cas surveillance complexes target double-stranded DNA, Type III complexes target single-stranded RNA. Near-atomic resolution cryo-electron microscopy (cryo-EM) reconstructions of native Type III Cmr (CRISPR RAMP module) complexes in the absence and presence of target RNA reveal a helical protein arrangement that positions the crRNA for substrate binding. Thumb-like β-hairpins intercalate between segments of duplexed crRNA:target RNA to facilitate cleavage of the target at 6-nt intervals. The Cmr complex is architecturally similar to the Type I CRISPR-Cascade complex, suggesting divergent evolution of these immune systems from a common ancestor.
Dr Raymond Staals is a Postdoctoral Fellow in the Department of Microbiology and Immunology at the University of Otago. The work was undertaken in collaboration with colleagues at his home university in Wageningen, Netherlands, along with The University of California, Berkeley.
Joshua P. Ramsay, Laura G. L. Tester, Anthony S. Major, John T. Sullivan, Christina D. Edgar, Torsten Kleffmann, Jackson R. Patterson-House, Drew A. Hall, Warren P. Tate, Michael F. Hynes, and Clive W. Ronson
PNAS 112 (13), pp 4104–4109.
Integrative and conjugative elements (ICEs) facilitate horizontal transfer of multiple genetic determinants. Here we show that a programmed ribosomal frameshift (PRF) contributes to the regulation of ICE transfer. The low-frequency PRF fuses the coding sequences of two genes, resulting in a single-protein Frameshifted excision activator (FseA) that activates ICE excision. An antiactivator, QseM, known to disrupt the quorum-sensing regulator TraR, also disrupted FseA. The evolved PRF site, together with the dual-target antiactivator, QseM, likely provides robust suppression of ICE transfer in the face of the inherent biological noise of quorum-sensing autoinduction. This work illustrates how a complex multipartite regulatory system has assembled through evolution to form a robust genetic toggle to control gene transcription and translation at both single-cell and cell-population levels.
Dr Josh Ramsay was a Health Sciences Career Development Postdoctoral Fellow in Prof Clive Ronson’s lab for the majority of this work; he is now a lecturer at Curtin University in Perth, WA. Laura Tester and Anthony Major were Masters students in Microbiology jointly supervised by Josh and Clive, Dr John Sullivan is an Assistant Research Fellow in Clive’s lab; Tina Edgar, Dr Torsten Kleffmann and Prof Warren Tate are collaborators in the Department of Biochemistry at Otago, Jackson Patterson-House and Drew Hall were Honours students with Josh at Curtin University, Prof Michael Hynes was a Visiting Professor in Clive’s lab from the University of Calgary in Canada.
Sergio E. Morales, Neha Jha, and Surinder Saggar
Soil Biology & Biochemistry, 82 (2015), 87e98
The results of this study indicate that, like plants and animals, microbial communities controlling emissions of the greenhouse gas nitrous oxide (N2O) are influenced by latitude. Although the mechanism is unclear, it suggests that soils closer to the poles have a higher, untapped potential for emitting greenhouse gases than those closer to the Equator. Under changing global conditions, this could result in higher gas emissions if temperature and moisture patterns shift.
Dr Sergio E. Morales is a Lecturer in the Department of Microbiology and Immunology. This work was undertaken in collaboration with Landcare Research and Massey University, part of the New Zealand Agricultural Greenhouse Gas Research Centre.
Htin Lin Aung, Debjit Dey, Peter H. Janssen, Ron S. Ronimus, Gregory M. Cook
Journal of Microbiological Methods, Volume 110, March 2015, Pages 15–17
The high-throughput screen developed in this study provides a biochemical platform to accelerate discovery of novel inhibitors of rumen methanogens to mitigate methane emissions.
Dr Htin Lin Aung is a Postdoctoral Fellow in Professor Gregory Cook's laboratory. This work was undertaken in collaboration with AgResearch, part of the Pastoral Greenhouse Gas Research Consortium. The PGgRc research programme aims to provide New Zealand livestock farmers with the knowledge and tools to mitigate greenhouse gas emissions from the agricultural sector.
Farah Al-Barwani, Sarah L. Young, Margaret A. Baird, David S. Larsen and Vernon K. Ward
PLoS ONE 9(8): e104523
Internalization of peptides by antigen presenting cells is crucial for the initiation of the adaptive immune response. Mannosylation has been demonstrated to enhance antigen uptake through mannose receptors, leading to improved immune responses. In this study we test the effect of surface mannosylation of protein-based virus-like particles (VLP) derived from Rabbit hemorrhagic disease virus (RHDV) on uptake by murine and human antigen presenting cells. A monomannoside and a novel dimannoside were synthesized and successfully conjugated to RHDV VLP capsid protein, providing approximately 270 mannose groups on the surface of each virus particle. VLP conjugated to the mannoside or dimannoside exhibited significantly enhanced binding and internalization by murine dendritic cells, macrophages and B cells as well as human dendritic cells and macrophages. This uptake was inhibited by the inclusion of mannan as a specific inhibitor of mannose specific uptake, demonstrating that mannosylation of VLP targets mannose receptor-based uptake. Consistent with mannose receptor-based uptake, partial retargeting of the intracellular processing of RHDV VLP was observed, confirming that mannosylation of VLP provides both enhanced uptake and modified processing of associated antigens.
Farah Al-Barwani is a PhD candidate in the Department of Microbiology and Immunology, working under the supervision of Professor Vernon Ward. The work was undertaken in collaboration with Sarah Young and Margaret Baird of the Department of Pathology and David Larsen from the Department of Chemistry.
Charlotte M. Wilson, Diane Loach, Blair Lawley, Tracey Bell, Ian M. Sims, Paul W. O'Toole, Aldert Zomer and Gerald W. Tannock
Applied and Environmental Microbiology, 2014, 80(19):6104
Habitat studies that measure bacterial functions can reveal mechanisms of ecological success of gut commensals, and their impact on host physiology. The study by Wilson et al. (AEM01876-14) showed that, while urease gene transcription was upregulated in vivo by L. reuteri as an acid tolerance mechanism, urea was metabolized to a greater extent in Lactobacillus-free mice compared to animals colonized by strain 100-23. This was because fecal-type bacteria in the stomach of Lactobacillus-free mice had high urease activity, but were suppressed by L. reuteri. This unexpected impact of lactobacilli on global urea hydrolysis highlighted the role of gut commensals on mammalian nitrogen recycling.
The article is based on some transcriptome experiments conducted by PhD student Charlotte Wilson, which led to further bacteriological and DNA-based studies, as well as collaborative work with chemists Drs Tracey Bell and Ian Sims at Callaghan Innovation. The article was selected by the editors of Applied and Environmental Microbiology for inclusion in "Spotlight," a feature in the Journal highlighting research articles in the upcoming issue that have been deemed of significant interest and includes short descriptions of four to six especially meritorious articles (reproduced below). The research was supported by the Marsden Fund.
Matloob Husain and Chen-Yi Cheung
Journal of Virology (2014)
Host cells produce many proteins that have the natural ability to restrict influenza A virus infection. In this paper, we have described a new class of host protein, histone deacetylase 6 (HDAC6) that inhibits the influenza A virus infection. We demonstrate that HDAC6 exerts its anti-influenza virus function by negatively regulating the trafficking of viral components to the site of influenza A virus assembly via its substrate acetylated microtubules. HDAC6 is a multi-substrate enzyme and regulates multiple pathways in the host cell, including the ones leading to various cancers, neurodegenerative diseases, and inflammatory disorders. Therefore, several drugs targeting HDAC6 are under clinical development for the treatment of wide range of diseases. Influenza A virus continues to be a major global public health problem due to regular emergence of drug-resistant and novel influenza A strains in humans. As an alternative antiviral strategy, HDAC6 modulators could be employed to stimulate the anti-influenza potential of endogenous HDAC6 to inhibit influenza A virus infection.
The research was conducted by Dr Matloob Husain and his technician Chen-Yi Cheung over the last two and half years, and supported by the Health Research Council, Otago School of Medical Sciences, and Department of Microbiology and Immunology.
*Richter, C., *Dy, R.L., *McKenzie, R.E., Watson, B.N.J., Taylor, C., Chang, J.T., McNeil, M.B., Staals, R.H.J. and Fineran, P.C.
Nucleic Acids Research (2014)
Journal impact factor = 8.28
The interactions between bacteria and their ‘parasites’, such as viruses and plasmids, underpin global nutrient cycles, the evolution of pathogens and antibiotic resistance. Bacteria and archaea protect themselves using an adaptive immune system, termed CRISPR-Cas, which has a sequence-specific genetic memory of previous invaders. This memory produces short interfering RNAs that specifically target and destroy invaders. Recently, CRISPR-Cas systems have revolutionised precision genome editing and have, for example, enabled the correction of genetic defects in adult mice. Despite this stunning technological advance, fundamental knowledge is lacking about how memories are derived from invaders. In this new study we show that the memory formation process is capable of rapidly eliciting new protective memories when facing invaders that were mutated following previous encounters. Exactly how these memories with partial recognition stimulate new memory formation is unknown, but our data have led us to propose a bi-directional translocation model for acquisition of new memories. Understanding these systems has broad implications for biotechnology and prokaryotic evolution.
The research was conducted by members of Peter Fineran’s lab over the last 2 years. Much of the work was the result of a recent Honours student (Becca McKenzie), a post-doc (Ron Dy) and a PhD student (Corinna Richter). Other authors include summer and MSc students, post-docs and a technician. The major support was from a Rutherford Discovery Fellowship and the University of Otago.
Anthea L. Bouwer, Sarah C. Saunderson, Felicity J. Caldwell, Tanvi T. Damani, Simon J. Pelham, Amy C. Dunn, Ralph W. Jack, Patrizia Stoitzner and Alexander D. McLellan
Journal of Immunology. 2014 Mar 1;192(5):2514-21.
Journal impact factor: 5.5
Recent research conducted by Associate Professor Alex McLellan and colleagues shows that bacteria may assist the body’s immune system response against cancer cells and help fight tumours like melanoma.
The article published in the official journal of the American Association of Immunologists, the Journal of Immunology, demonstrates that bacteria stimulate a type of immune response that results in more effective killer cell attacks against cancer.
Increasing evidence suggests that NK cells act to promote effective T cell–based antitumor responses. Using the B16-OVA melanoma model and an optimized Gram-positive bacteria–dendritic cell (DC) vaccination strategy, we determined that in vivo depletion of NK cells at time of tumor challenge abolished the benefit of DC immunotherapy. The contribution of NK cells to DC immunotherapy was dependent on tumor Ag presentation by DC, suggesting that NK cells act as helper cells to prime or reactivate tumor-specific T cells. The absence of NK cells at tumor challenge resulted in greater attenuation of tumor immunity than observed with selective depletion of either CD4 or CD8 T cell subsets. Although successful DC immunotherapy required IFN-γ, perforin expression was dispensable. Closer examination of the role of NK cells as helper cells in enhancing antitumor responses will reveal new strategies for clinical interventions using DC-based immunotherapy.